Assessment of RNA integrity as sample quality control is critical for good microarray performance and analysis. RNA degradation is common, for example in samples from tissue biopsies where there is often considerable delay during surgery before appropriate storage. In RNA integrity quality control, commonly the ratio of the two major ribosomal RNAs (rRNA) is used to indicate general RNA integrity quality (typically 18S and 28S in eukaryotes, 16S and 23S in prokaryotes). These are abundant transcripts (over half the total cellular RNA) and easily resolved by agarose gel or microfluidic systems. Typically a 2:1 ratio in the total amount of the 28S rRNA compared to the 18S rRNA indicates a high integrity sample.
Typical disadvantages of pre-analysis RNA quality control by e.g. gel electrophoresis are the requirement of high amounts of total RNA (approximately 1 μg) and often the requirement of a substantial amount of the sample to be expended in this assessment.
In current practices, however, a growing requirement is to assess RNA quality in small (picogram to nanogram) samples—for example those obtained from laser capture micro dissection and often, current methods requiring the entire sample or more to be expended in pre-analysis quality control are therefore not appropriate.
Efforts to reduce the amount of total RNA for quality assessment have been numerous including RT-PCR of the 18S rRNA using 25 ng or less starting material but this is a significant effort. Alternatively, hybridisation of primers to the 28S rRNA have been used as a predictor of RNA integrity in in situ hybridisations.
Further minimalisation of sample quantity was achieved by the introduction of Agilent's Lab-on-a-Chip technology which provides an alternative to traditional gel-based analysis that integrates the quantitation of RNA samples with quality assessment in a single assay; as little as 1 μl of 10 ng/μl is required per analysis.
The challenge to perform an integrated microarray analysis of RNA-derived samples, including sample analysis (e.g. expression profiling) as well as sample quality/integrity assessment, however, remains.
Various scenarios of microarray analysis quality control exist; employing an internal standard which may be exogenous or endogenous. Current quality control, however merely focuses on the normalization of the output results to compensate for differences in labelling and detection efficiencies and for differences in spotted nucleic acid quantity, surface anomalies and overall slide quality. RNA integrity in current microarray analysis is not addressed.
The feasibility of using rRNA as an endogenous standard in microarray analysis has been demonstrated in WO 02/05038 which discloses a method for providing an rRNA internal standard.
rRNAs as an internal endogenous standard is generally considered ideal in a large experimental field in view of its expression which does not vary during cell cycle, between cell types, or in response to the experimental treatments that one wishes to examine. However, for such an endogenous standard to be valid in nucleic acid microarray analysis it is crucial that it be of similar relative abundance as the target transcript. Since rRNA is found in massive amounts relative to other RNAs, any simultaneous method of detecting rRNAs at the same time as other RNA species must take this into account (e.g. by differential amplification) while remaining an accurate assessment of the original levels.
In WO 02/05038, the excess signal coming from the labelled rRNA (or from the cDNA generated from the rRNA) is competed out and the signal detected for it is reduced to a range compatible with that of the signal for the other labelled RNA's by the post-transcriptional addition of a quantity of an unlabelled ribosomal nucleic acid competitor probe into the hybridisation buffer to compete with the immobilized ribosomal capture probe. The use of such internal standard in test and reference samples then allows for the provision of a normalization factor which allows for the correction of the hybridisation signal resulting from the binding of target cDNA with corresponding capture probe. A problem with the above method is however that a lot of excess of fluorescently labelled amplified rRNA is present in the hybridisation mix, which contributes to high background signals.
In current practice, however no microarray hybridisation analysis with integrated RNA integrity control has yet been proved feasible. There is a need for the assessment of RNA integrity to be related to the performance of hybridisations of the amplified RNA (aRNA) to the array.
The present invention aims at providing a method to assess RNA integrity in microarray analysis assays for nucleic acid hybridisation. More in particular, the present invention aims at providing a method for microarray analysis of a nucleic acid sample wherein said sample is analysed for its original integrity in combination with its analysis towards a particular application; e.g. gene expression profiling. The method of the present invention thus aims at providing a nucleic acid analysis method with no requirement to expend any sample to a pre-analysis quality control.